Introduction

Diversity

1424-2818

Molecular Diversity Preservation International (MDPI)

10.3390/d3040547

diversity-03-00547

Article

Diversity of Pharmacological Properties in Chinese and European Medicinal Plants: Cytotoxicity, Antiviral and Antitrypanosomal Screening of 82 Herbal Drugs

Herrmann

Florian

¹ Romero

Marta R.

² Blazquez

Alba G.

² Kaufmann

Dorothea

¹ Ashour

Mohamed L.

³ Kahl

Stefan

⁴ Marin

Jose J.G.

² Efferth

Thomas

⁵ Wink

Michael

¹^*

1 Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Heidelberg 69120, Germany; E-Mails: Florian.Herrmann@t-online.de (F.H.); D.Kaufmann@uni-heidelberg.de (D.K.) 2 Laboratory of Experimental Hepatology and Drug Targeting (HEVEFARM), National Institute for the Study of Liver and Gastrointestinal Diseases (CIBERehd), University of Salamanca, Salamanca 37007, Spain; E-Mails: marta.rodriguez@ciberehd.org (M.R.R.); albamgb@usal.es (A.G.B.); jjgmarin@usal.es (J.J.G.M.) 3 Department of Pharmacognosy, Faculty of Pharmacy, Ain Shams University, Abbassia, Cairo 11566, Egypt; E-Mail: Mohamed_ashour@pharm.asu.edu.eg (M.L.A.) 4 Isostatic Products, RHI AG, Leoben 8700, Austria; E-Mail: Stefan.kahl@gmail.com 5 Department of Pharmaceutical Biology, Institute of Pharmacy and Biochemistry, University of Mainz, Mainz 55128, Germany; E-Mail: efferth@uni-mainz.de

* Author to whom correspondence should be addressed; E-Mail: wink@uni-hd.de; Tel.: +49-6221-54-4880; Fax: +49-6221-54-4884

2011

26 09 2011

3 4 547 580 29 07 2011 05 08 2011 15 09 2011

2011

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).

In an extensive screening, the antiviral, antitrypanosomal and anticancer properties of extracts from 82 plants used in traditional Chinese medicine and European phytomedicine were determined. Several promising plants that were highly effective against hepatitis B virus (HBV), bovine viral diarrhoea virus (BVDV)—a flavivirus used here as a surrogate in vitro model of hepatitis C virus, trypanosomes (Trypanosoma brucei brucei) and several cancer cell lines were identified. Six aqueous extracts from Celosia cristata, Ophioglossum vulgatum, Houttuynia cordata, Selaginella tamariscina, Alpinia galanga and Alpinia oxyphylla showed significant antiviral effects against BVDV without toxic effects on host embryonic bovine trachea (EBTr) cells, while Evodia lepta, Hedyotis diffusa and Glycyrrhiza spp. demonstrated promising activities against the HBV without toxic effects on host human hepatoblastoma cells transfected with HBV-DNA (HepG2 2.2.15) cells. Seven organic extracts from Alpinia oxyphylla, Coptis chinensis, Kadsura longipedunculata, Arctium lappa, Panax ginseng, Panax notoginseng and Saposhnikovia divaricata inhibited T. b. brucei. Moreover, among fifteen water extracts that combined high antiproliferative activity (IC₅₀ 0.5–20 μg/mL) and low acute in vitro toxicity (0–10% reduction in cell viability at IC₅₀), Coptis chinensis presented the best beneficial characteristics. In conclusion, traditional herbal medicine from Europe and China still has a potential for new therapeutic targets and therapeutic applications.

anticancer drugs gastrointestinal tumors traditional Chinese medicine cytotoxicity hepatitis B hepatitis C HepG2 2.2.15 BVDV Trypanosoma brucei brucei

1. Introduction

Traditional Chinese medicine (TCM) has a long history starting with the Shang Dynasty around 1500 BC and officially uses approximately 4773 herbs, while the number of locally used plants is probably much higher [1]. Clinical efficacy was shown in various examples, one of the best known is that of artemisinin from Artemisia annua, commonly used against malaria, but also effective against T. b. brucei, viral infections and cancer [2-8].

European medicine also has a long tradition of at least 2500 years with the two important early scholars Hippocrates and Dioscorides who described more than 400 medicinal plants 2000 years ago, many of which are still in use today [9]. Many pure therapeutic agents used in modern medicine were originally based on herbal medicine; in fact, the process of developing new drugs from European herbal medicine is still alive and important discoveries are regularly made [10,11]. Even though the theoretical concept of traditional medicine differs between Europe and China, often the same plants were and are still used in both cultures to treat the same or similar health disorders. Modern European phytotherapy also includes important herbal medicines from Africa and America.

Even though the diversity of plants and possible natural products is vast, the number of targets is usually limited (Table 1). Most natural products target proteins, biomembranes or DNA unselectively. Selective interaction is often the case when especially alkaloids mimic signal molecules and interact with receptors or enzymes. It is often possible to conclude from the type of the natural products to their most likely mode of action. Saponins and monoterpenes are active on the biomembrane, while polyphenols usually interact with proteins. Alkaloids also interact with proteins or the DNA.

The formations of covalent and of non-covalent bonds are the two modes of action that form the basis of all interactions between proteins and natural products.

The two main targets for the formation of covalent bonds are free amino and free SH groups. Aldehydes, isothiocyanates and epoxids can form covalent bonds with free amino groups while sesquiterpene lactones, disulfides (e.g., allicin), polyacetylenes and epoxides can form covalent bonds with free SH groups.

The second mechanism of maybe even greater importance due to its universality is the formation of non-covalent bonds between phenolic OH-groups and amino groups. The proton of the phenolic OH-group can partly dissociate under physiological conditions so that unspecific interactions by forming strong, ionic bonds occur with proteins. Tannins are especially effective due to their large number of hydroxyl groups.

All of these interactions will change the three dimensional structure of the protein and thus inactivate it. The omnipresence of these unspecific natural products in plants explains the efficacy of many plant extracts. They are responsible for the great number of “hits” usually occurring in extended screenings of medicinal plant extracts (Table 2).

Hepatitis B and hepatitis C are responsible for 75% of all cases of liver diseases worldwide, often causing cirrhosis and hepatocellular carcinoma [14,15]. Hepatitis B and hepatitis C account for the most problematic viral infections, since the standard treatment with pegylated IFN-γ and the purine nucleoside analogues lamivudine and ribavirin have severe side effects while being at the same time ineffective for 50% of the patients [14,16]. Thus, new drugs are urgently needed [17]. Together with the bovine viral diarrhoea virus (BVDV), and the Japanese Encephalitis virus, hepatitis C virus (HCV) belongs to the Flaviviridae family. As BVDV, whose cytopathic strains induce a lytic infection in some cell lines, such as embryonic bovine trachea (EBTr) cells, is easier to manipulate and lacks human infectivity, this is commonly used as in vitro model for infections of this viral family [18].

Our knowledge of the natural products of many plants used in European and Chinese phytomedicine is broad (Table 2), however, many new discoveries are still possible. Previously, several studies demonstrated the promising potential of traditional phytomedicine for the discovery of new antiviral drugs. Artemisinin and related compounds proved effective in screening assays against viral hepatitis [6,7,19]. In water extracts of Terminalia chebula, Sanguisorba officinalis, Rubus coreanus and Rheum palmatum, Kim et al. [20] discovered prominent anti-hepatitis B virus (HBV) activities. The ethanolic extract of Hypericum perforatum, a well-established drug for treatment of depression [9] was also shown to be active against the HBV [21]. Laxative anthraquinones isolated from Rheum palmatum demonstrated significant effects against HBV [22] and saikosaponins from Bupleurum species were previously shown to lower significantly the HBV level in the HepG2 2.2.15 assay [23]. HepG2 2.2.15 is a stable cell line infected with the HBV. The assay measures the production of secreted HBV from the cell by using real time quantitative PCR.

Parasites such as protozoa and helminths cause a major health threat in many tropical countries [24], while suitable drugs are still rare [25]. Blood parasites of the genus Trypanosoma (Trypanosoma brucei rhodesiense and T. b. gambiense) are responsible for African trypanosomiasis (sleeping sickness) with serious consequences for human health and economy. Due to the high infectivity of African human trypanosomes, T. b. brucei is commonly used as model organism with similar morphology and biochemical processes, while being only infective for cattle [24,26,27]. This subspecies causes the cattle epidemic nagana, it is responsible for severe financial loss of 1340 billion USD per year [28].

Currently, only four drugs are approved internationally for the treatment of humans against sleeping sickness: suramin, pentamidine, melarsoprol and eflornithine. Diminazene, another effective antitrypanosomal drug, is only approved for the use on animals because of severe side effects [24]. Even the drugs approved for human use are responsible for serious side effects, and furthermore, the parasites develop increasing resistance to them [29-32]. This situation makes the discovery of new, effective drugs an urgent task of the 21st century [33-35].

When considered together, enterohepatic tumors, i.e., those affecting the liver, the biliary duct, gallbladder and the intestine, constitute the first cause of death due to cancer. Although in many cases surgery and radiotherapy are efficacious, these therapeutic strategies cannot always be applied. Moreover, even when the removal of tumors is possible, pre- and post-operative pharmacological adjuvant regimens are often needed. However, one important limitation to the use of cytostatic drugs to treat enterohepatic tumors is that they generally exhibit marked resistance to currently available pharmacological approaches and the development of resistance during treatment [36].

Many natural products and derivatives thereof belong to the standard repertoire of cancer chemotherapy. Examples are Vinca alkaloids, such as vincristine, vinblastine and vinorelbine, obtained from Madagascar periwinkle (Catharanthus rosea). Also taxanes such as paclitaxel and docetaxel, which are produced from the bark of Pacific yew (Taxus), podophyllotoxins, such as etoposide and teniposide, derivatives of the genus Podophyllum, and camptothecin, derived from the Asian “Tree of Happiness” (Camptotheca acuminata) and its derivatives, irinotecan and topotecan, are natural products from TCM plants [4].

In this study, extracts from 82 traditional medicinal plants were screened against HBV and flaviviruses, T. b. brucei and several cancer cell lines. Our aim was to detect new sources of active compounds for the possible treatment of these important causes of diseases.

2. Experimental Section 2.1. Chemicals

Dimethylsulfoxide (DMSO), trypsin-EDTA, DMEM and MEM with GLUTAMAX media, fetal bovine serum (FBS) and supplementary chemicals were bought from Gibco^® Invitrogen; Germany. Antibiotic/antimycotic solution, gentamicin, Neutral Red (NR, 3-amino-7-dimethylamino-2-methylfenazine), NaHCO₃, L-glutamine and MEM media were purchased from Sigma-Aldrich (Madrid, Spain). Geneticin^® (G418) was from Roche (Barcelona, Spain). Dried TCM plants were obtained in Shanghai; South African plants were provided by Prof. van Wyk, University of Johannesburg, South Africa.

2.2. Authentication of Plant Material

The TCM plants were genetically identified by DNA barcoding to confirm the identity and to exclude adulterations. DNA was isolated from plant drugs; their chloroplast rbcL gene was amplified and sequenced. The obtained sequences were authenticated with sequences obtained from sample species of the Botanical Garden of Heidelberg and databases. Voucher specimens of the plant material were deposited at the Department of Biology, Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Germany. Additionally, the plants were authenticated by visual and microscopic techniques.

2.3. Extract Preparation

Five hundred grams of dry plant material was powdered and extracted with dichloromethane, methanol and water under moderate heat using a reflux condenser for 4 hours. The extracts obtained were concentrated using the rotation evaporator, stored at −40 °C under exclusion of light and dried under vacuum prior to the experiments. Dried extracts were dissolved in DMSO for the experiments.

2.4. Test Organisms

T. b. brucei TC 221 were originally obtained from Prof. Peter Overath (Max-Plank Institut für Biologie, Tübingen) by Dr. D. Steverding before being cultured at the IPMB, Heidelberg since 1999. HeLa cancer cells and Cos7 fibroblast cells (African green monkey kidney cells immortalized with the monkey virus SV40) were cultured at the IPMB, Heidelberg for several years; Hep G2, SK-Hep1 and LS 174T, HepG2 2.2.15 and EBTr cells were cultured at the Laboratory of Experimental Hepatology and Drug Targeting (HEVEFARM), University of Salamanca, CIBERehd, Spain.

2.5. Methods

Cancer Cells (HeLa, Hep G2, SK-Hep1 and LS 174 T) were basically grown as previously described [37], HeLa and Cos7 cells were grown at 37 °C with 5% CO₂ in DMEM complete media (10% heat-inactivated FBS; 5% penicillin/streptomycin; 5% non-essential amino acids). Hep G2, SK-Hep1, and LS 174T cells were grown at 37 °C with 5% CO₂ in MEM complete media (10% heat-inactivated FBS; 1% antibiotic-antimycotic solution).

HepG 2.2.15 cells were cultured as previously described [7] in DMEM complete medium with 10% FBS, geneticin and gentamicin. EBTr cells were cultured as described elsewhere [6], they were maintained in MEM-GLUTAMAX medium with 10% heat-inactivated FBS; 1% penicillin/streptomycin, and 0.1% gentamicin.

T. b. brucei TC221 cells were cultured in BALTZ medium [38] supplemented with 20% inactivated FBS and 0.001% β-mercaptoethanol.

The MTT cell viability assay was used to determine cytotoxicity in Cos7 and HeLa cells [39,40]. Cells during the logarithmic growth period were seeded in 96 well plates (Greiner Labortechnik) at concentrations of 2 × 10⁴ cells/well and grown for 24 h. Dried and powdered extracts were dissolved in DMSO before being serially diluted to 10 concentrations in 96 well plates. Cells were incubated with the extract for 24 h before the medium was removed and replaces with fresh medium containing 0.5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The formazan crystals were dissolved in DMSO 4 h later; the absorbance was measured at 570 nm with a Tecan Safire II Reader.

T. b. brucei TC221 cell viability was additionally to the MTT assay confirmed and evaluated using microscopic techniques.

Toxicity of the extracts for T. b. brucei was compared to HeLa and Cos7 cells and the Selectivity index (SI) was calculated. SI: ratio of the IC₅₀ value of mammalian cells divided by the IC₅₀ value of trypanosomes.

To test the antiproliferative effect, 5 × 10³ or 15 × 10³ cells per well (depending on the cell line) were seeded in 96 well plates and incubated with 5, 10, 25, 50, 100, and 200 μg/mL water extract for 72 h. The cell viability was also determined using the MTT assay with minor modifications. Acute toxicity was similarly measured using MTT assay but after short-term (6 h) incubation with the extracts at the concentrations of IC₅₀ calculated for each cell line.

To determine the antiviral effect of the extracts, BVDV was used here as a substitute in vitro model for HCV infection. Bovine epithelial cells obtained from embryonic trachea (EBTr) were cultured in MEM with GLUTAMAX medium as described previously [6]. They were seeded in 96 well plates (15 × 10³ cells/well; 50 μL/well) and left to attach for 2 h. Afterwards, the cells were infected with 50 μL/well of the desired dilution in culture medium of an initial suspension of BVDV (cytopathic strain Oregon C24V, genotype I, subgenotype b) to reach 40% cytopathic effect. After 48 h of incubation the medium was replaced with dilutions in culture medium of the extracts (1, 5, 10, 50, 100 μg/mL). The viability of the EBTr cells was measured using the MTT assay after 72 h incubation.

An HBV antiviral assay based on the HepG2 2.2.15 model was used to determine the antiviral activity of the extracts [41]. HepG2 2.2.15 cells were seeded in six-wells plates (35 × 10⁴ per well) before being incubated for 21 days with 50 μg/mL, 25 μg/mL and 12.5 μg/mL extract. The culture medium was replaced every 3 days with fresh medium, containing the extract dilutions. Quantitative real-time PCR (QPCR) was used to measure the HBV-DNA levels in the culture medium (representing HBV virion production) as described previously [7]. Cytotoxicity was determined using the uptake of NR dye at the end of treatment [42].

At least three cultures for each experimental condition were carried out. Data points were obtained in triplicate form (T. b. brucei, cancer cell lines, Cos7, HepG2 2.2.15 cells) and in 8 different wells (EBTr). The IC₅₀ value was calculated using SigmaPlot^® 11.0 (4 parameter logistic curve). Statistical significance determined via paired t-test or the Bonferroni method of multiple-range testing.

3. Results and Discussion

The great diversity of natural products occurring plants is of the utmost importance for the discovery of new pharmaceutical lead compounds. Through millions of years of evolution the defence mechanisms of plants were perfected. The great variety of natural products clearly demonstrates the efficacy of this defence strategy against herbivores, but also fungi, bacteria and viruses (Table 2). In many cases the plants do not rely on specific interactions but also rely on unspecific molecules that interact with a great number of targets (Table 1). Of highest importance are the interactions with free amino and free SH groups. While aldehydes, isothiocyanates and epoxids are able to form covalent bonds with free amino groups, sesquiterpene lactones, disulfides, polyacetylenes and epoxides interact with free SH groups. Phenolic OH-groups interact on a non-covalent basis with free amino groups by forming strong hydrogen and ionic bonds.

The cytotoxicity of water and organic solvent extracts from 82 medicinal plants was determined in the fibroblast cells Cos7 and in four cancer cell lines: HeLa, HepG2, SK-Hep1 and LS 174T (Tables 3 and 4). The aqueous extracts were also screened against BVDV and HBV (Table 3), whereas organic solvent extracts were assayed on T. b. brucei (Table 4). Our results revealed promising results in order to use several of these plants as sources for therapeutic agents.

The viral particles offer three main targets to the natural products (Table 1). They can interact with the surface proteins, the biomembrane and the DNA or RNA. While most plants interact unselectively with the virus, selective interactions do also occur.

10 plants demonstrated antiviral protection against BVDV in combination with low cytotoxicity. Four plants (Panax ginseng, Cassia tora, Ginkgo biloba and Viola yezoensis) exerted protective antiviral effect only at high doses, whereas other six plant extracts (Celosia cristata, Ophioglossum vulgatum, Houttuynia cordata, Selaginella tamariscina, Alpinia galanga and Alpinia oxyphylla) were effective at lower concentrations (Table 3).

Regarding the six plants with higher potential interest as a source of anti-HCV drugs, antiviral glycoproteins, CCP-25 and CCP-27, purified from the leaves of Celosia cristata [43] have been previously studied [44-48]. Their ability to inhibit viral RNA translation activities against several plant viruses have been described [49].

Quercetin 3-O-methyl ether and ophioglonin obtained from plants belonging to Ophioglossaceae genus have shown slight activity against HBV [50]. Since 1995 when antiviral activities against enveloped viruses were discovered in extracts of Houttuynia cordata [51], such as influenza, HIV, herpes, SARS and also in enteroviruses [51-54], 40 compounds have been isolated from the whole plant [55].

Among all of them, norcepharadione B has been identified as anti-herpes virus type 1 compound [55], quercetin may reduce virions production of HCV [56], but not against HBV [7] and quercetin 3-rhamnoside may be effective against influenza A virus [57].

Selaginella tamariscina has been a source of several drugs with anti-bacterial and antifungal activities such as amentoflavone [58], isocryptomerin [59-61], or with antitumor effects such as sterols [62] and biflavonoids [63]. Alpinia galanga crude extracts have been shown to have antibacterial activities [64] which seem to be enhanced in combination with other plants such as rosemary and lemon iron bark [65]. Compounds obtained from this plant, have also demonstrated other antimicrobial activities, such as anti-leishmanial phenylpropanoids [66] or 1′-acetoxychavicol acetate, and its halogenated derivatives (inhibitors of HIV-regulator protein Rev-export) [67-70].

The insecticidal properties of diarylheptanoid [71] as well as protective effects on anaphylactic reactions of the aqueous extracts from the fruit of Alpinia oxyphylla [72] have been described in the past. Recently anti-angiogenic properties of the fruit have been also discovered [73].

The water extracts were also screened against HBV in HepG2 2.2.15 cells (Table 3). Evodia lepta, Hedyotis diffusa and several Glycyrrhiza species lowered the HBV-DNA significantly and were not toxic to the HepG2 2.2.15 cell line (Figure 1).

Hardly anything is known about the other natural products of Evodia lepta, while the highly bioactive chromenes seem to be among the major constituents [74]. Glycyrrhiza species, on the other hand, are well known for their anti-inflammatory effects due to glycyrrhizic acid [9]. This genus is also known for its antiviral, especially antihepatitis properties [15,75]. Its ability to reduce the HBV-DNA in the culture medium of HepG2 2.2.15 at high doses has been previously reported [7]. Heydiotis diffusa again is a plant rich in iridoid glycosides with anti-inflammatory and hepatoprotective activities [76-78]. These compounds are most likely to be responsible for the effects against HBV.

Three enterohepatic cancer cell lines, HepG2 and SK-Hep1 (from human hepatoblastoma and hepatocarcinoma) and LS 174T (from human colon adenocarcinoma), were used to determine the antitumor ability of water extracts (Table 3). Twenty extracts were found to induce a significant antiproliferative effect with IC₅₀ values between 0.5 and 20 μg/mL on these cell lines. These were further investigated to elucidate whether this was due to cytotoxicity.

In HepG2, none of the 4 extracts with ability to inhibit cell growth (Coptis chinensis, Epimedium brevicornum, Equisetum hiemale and Senecio scandens), were found to induce acute cell toxicity when they were incubated with the IC₅₀ of the extracts for 6 h (Figure 2).

In SK-Hep1, among the 10 extracts with antitumor effect 7 did not induce acute toxicity (Arctium lappa, Cassia tora, Centipeda minima, Chrysanthemum indicum, Coptis chinensis, Phellodendron chinense and Rheum palmatum), whereas Dysosma versipellis was especially active by lowering the cell viability in comparison to the control to 40% (Figure 3). This is consistent with the inhibitory effects known for the lignans of D. versipellis against prostate cancer cell lines [79].

Evodia lepta and Kadsura longipedunculata lowered also the cell viability of SK-Hep1 in comparison to the control to 50-60%. Recently, it has been reported that the essential oil of Kadsura longipedunculata and its major components (delta-cadinene, camphene, borneol, cubenol, and delta-cadinol) have some degree of cytotoxic activity against some human cell lines [80]. In LS 174T cells, water extracts from Coptis chinensis, Dysosma versipellis, Epimedium brevicornum, Hedyotis diffusa and Houttuynia cordata have antiproliferative effects without affecting cell viability (Figure 4), whereas Paeonia lactiflora, Platycladus orientalis, and Polygonum aviculare, in addition to inhibition of cell growth were able to acutely lower cell viability in comparison to the control to 60–70%.

Paeonia lactiflora, which belongs to the Paeoniaceae family, is known as one of the richest sources of various resveratrol derivatives [81]. These phytoestrogens are known to exert strong antioxidant activity [81] and to inhibit growth of several cancer cell lines [82,83], including a colon human cell line [84]. Recently, the antiproliferative effects of essential oils obtained from Platycladus orientalis on human renal adenocarcinoma and amelanotic melanoma cells have been reported [85].

Coptis chinensis, which has been found active against the three enterohepatic cell lines, belongs to TCM formulations commonly used to treat liver diseases associated to infections by gastrointestinal parasite such as Blastocystis hominis [86]. Coptisine, which is used as gastric mucosa protector, and berberine, which has very interesting properties as antiinflamatory, antidiabetes, antidiarrhea, and hypocholesterolemic drug, have been obtained from this plant. Both of them have also shown antitumoral activities in in vitro models [87-92].

Animal cells offer several targets to natural products (Table 1). Of great importance are the biomembrane, the proteins and the DNA. Since human cells and trypanosomes share many similarities in the structure of the cells, it is extremely important to select those plant extracts where a great selectivity index (SI) occurred. The different cytotoxicity strongly hints to selective interactions between natural products and trypanosomes that do not occur in human cells. A great SI also hints to the relative absence or relative insignificance of general cytotoxic mechanisms like the unspecific interaction of phenolic OH-groups compared to more specific interactions with certain structures in trypanosomes.

The CH₂Cl₂ and MeOH extracts of 82 medicinal plants were screened against the cell lines HeLa, Cos7 and trypanosomes (T. b. brucei) (Table 4). The SI of the IC₅₀ of mammalian cell/trypanosomes was regarded as significant if it was over 80. According to this criterium, seven extracts were highly selective towards trypanosomes.

The CH₂Cl₂ extract of Alpinia oxyphylla showed IC₅₀ values of 119.6 μg/mL, 30.4 μg/mL and 0.7 μg/mL against HeLa, Cos7 and T. b. brucei respectively with SI of 170 and 43 between HeLa and T. b. brucei and Cos7 and T. b. brucei respectively. The MeOH extract of A. oxyphylla also was effective with IC₅₀ values of 213.8 μg/mL, 110.2 μg/mL and 2.0 μg/mL against HeLa, Cos7 and T. b. brucei respectively. The SI was 107 and 55 between HeLa and T. b. brucei and Cos7 and T. b. brucei respectively. A. oxyphylla is basically an essential oil plant, so that we suspect the active principle to be based on the sesquiterpenes already known for their cytotoxic properties [93].

For Kadsura longipedunculata only the CH₂Cl₂ extract exhibited a significant selectivity. Here, the IC₅₀ values of HeLa, Cos7 and T. b. brucei were 9.9 μg/mL, 1.8 μg/mL and 0.1 μg/mL respectively, resulting in SI of 99 and 18 between HeLa and T. b. brucei and Cos7 and T. b. brucei respectively. Essential oil and lignans form the major natural compounds of K. longipedunculata [80,94]. The specific trypanocidal effect rather seems to be based on the lignans than on the more unspecific essential oil. Further studies would be necessary to confirm this assumption.

For Arctium lappa only the CH₂Cl₂ extract showed a significant selectivity with IC₅₀ values of 345.0 μg/mL, 344.2 μg/mL and 3.6 μg/mL against HeLa, Cos7 and T. b. brucei respectively and SI of 96 between HeLa and T. b. brucei and Cos7 and T. b. brucei.

In Panax ginseng and P. notoginseng the selectivity was again limited to the CH₂Cl₂ extract. P. ginseng gave IC₅₀ values of 152.4 μg/mL, 47.7 μg/mL and 0.9 μg/mL against HeLa, Cos7 and T. b. brucei respectively with SI of 169 and 53 between HeLa and T. b. brucei and Cos7 and T. b. brucei respectively. P. notoginseng demonstrated IC₅₀ values of 263.0 μg/mL, 6.4 μg/mL and 0.9 μg/mL with SI of 292 and 7 between HeLa and T. b. brucei and Cos7 and T. b. brucei respectively.

Also an extremely active plant was Saposhnikovia divaricata. Here as well, lipophilic CH₂Cl₂ extract was selective with IC₅₀ values of 410.1 μg/mL, 45.9 μg/mL and 5.1 μg/mL against HeLa, Cos7 and T. b. brucei respectively and SI of 80 and 9 between HeLa and T. b. brucei and Cos7 and T. b. brucei respectively.

The trypanocidal effects of A. lappa, P. ginseng, P.notoginseng and S. divaricata are based on the presence of highly reactive polyacetylenes, especially panaxynol.

Only the methanolic extract of Coptis chinensis showed a significant selectivity, but not the dichloromethane extract. The IC₅₀ values of 81.8 μg/mL, 3.7 μg/mL and 0.4 μg/mL against HeLa, Cos7 and T. b. brucei respectively gave SI of 205 and 9 between HeLa and T. b. brucei and Cos7 and T. b. brucei respectively. Our analytical data confirmed berberine as the main alkaloid of C. chinensis. The toxicity of C. chinensis is probably an effect of the DNA intercalation of its alkaloids into the DNA double helix of T. b. brucei [95,96].

The trypanocidal effect of berberine against different trypanosoma species has been demonstrated previously. Merschjohann et al. [97] showed that T. congolense are sensitive to berberine at concentrations of 83 μM, while Rosenkranz and Wink [98] demonstrated a sensitivity of T. brucei to berberine at concentrations of only 0.5 μM. Recently, the effect of berberine against T. rhodesiense was also established by Freiburghaus et al. [99]. T. rhodesiense was sensitive to 4.2 μg/mL.

The significant differences in sensitivity of different trypanosoma species to berberine could be of high interest regarding resistance mechanisms against mutagenic compounds. Berberine might due to its mutagenic activity never become a lead structure for the development of trypanocidal drugs, but the differences in sensitivity of these three trypanosoma species might help to understand defence mechanisms against DNA intercalating substances.

4. Conclusions

Traditional Chinese and European Medicine comprise promising plants that might be used for antiviral, antitrypanosomal and anticancer therapy. The promising discoveries of highly effective plants against viral hepatitis, trypanosomiasis and liver and intestinal cancer cells, however, require further research to establish new lead structures or their combinations for the treatment of these important traditional diseases.

Figures and Tables Figure 1.

Effect of water extracts on hepatitis B virus (HBV) release as determined by HBV-DNA content in the culture medium and cell viability as determined by Neutral Red uptake by human hepatoblastoma cells HepG2 2.2.15 infected with HBV. Values are means ± SD of three experiments carried out in triplicate by incubation with the extracts for 21 days. *, p < 0.05 as compared with untreated cells by paired t-test.

Figure 2.

Acute cell toxicity as determined by 3-(4,5-dimethylthiazol-2-yl)-2, 5-difenyltetrazolium (MTT) assay in human hepatoblastoma HepG2 cells. Values are means ± SD of four experiments carried out in triplicate.

Figure 3.

Acute cell toxicity as determined by MTT assay in human hepatoma SK-Hep1 cells. Values are means ± SD of four experiments carried out in triplicate. *, p < 0.05 as compared with Control by the Bonferroni method of multiple range testing.

Figure 4.

Acute cell toxicity as determined by MTT assay in human colon adenocarcinoma LS 174T cells. Values are means ± SD of four experiments carried out in triplicate. *, p < 0.05 as compared with Control by the Bonferroni method of multiple range testing.

Table 1.

Targets in animal cells, bacteria cells and viruses [12].

Target	Activity	Secondary metabolites
Biomembrane
	Membrane disruption	Saponins
	Disturbance of membrane fluidity	Saponins, monoterpenes
	Disturbance of membrane proteins	Monoterpenes

Proteins (unspecific interaction)
	Non-covalent bonding (change of 3D protein conformation)	Phenolic molecules (flavonoids, catechins, tannins, anthraquinones, quinones, lignans, phenylpropanoids)
	Covalent bonding (change of 3D protein conformation)	Allicin, furanocoumarins, isothiocyanates, sesquiterpene lactones, aldehydes, epoxids, triple bonds

Proteins (specific interaction)
	Inhibition of enzymes	Structural mimetics of signal molecules (many alkaloids, e.g., nicotine), hydrogen cyanide from cyanogens
	Inhibition of Na⁺ K⁺ pumps	Cardiac glycosides
	Inhibition of microtubule formation	Colchicine, podophyllotoxin, taxol, vinblastine
	Inhibition of protein biosynthesis	Emetine, lectins
	Inhibition of transporters	Non-protein amino acids
	Modulation of hormone receptors	Isoflavonoids
	Modulation of ion channels	Many alkaloids, aconitine
	Modulation of neuroreceptors	Many alkaloids, some non-protein amino acids
	Modulation of regulatory proteins	Caffeine, phorbol esters
	Modulation of transcription factors	Structural mimetics of hormones (e.g., isoflavones genistein, daidzein)

DNA/RNA
	Covalent modification (alkylation)	Aristolochic acids, furanocoumarins, pyrrolizidine alkaloids, molecules with epoxy groups
	Inhibition of DNA topoisomerase I	Berberine, camptothecin
	Inhibition of transcription	Amanitine
	Intercalation	Planar, aromatic and lipophilic molecules (anthraquinones, berberine, emetine, quinine, sanguinarine, furanocoumarins)

Table 2.

Main Compounds of Plants used in this study [9,13].

Family	Species	Main Compounds
Acanthaceae	Andrographis paniculata	Diterpenelactones

Amaranthaceae	Celosia cristata	Lectins (amarathin, isoamaranthin, celosianin), ferulic acid

Apiaceae	Bupleurum chinense	Flavonoids (quercetin, rutin, isoquercetin, isorhamnetin), β-sitosterol, β-sitosterol-3-O-glucosid, α-spinasterol, α-spinasterol-3-O-glucoside
	Bupleurum marginatum	Flavonoids (quercetin, rutin, isoquercetin, isorhamnetin), β-sitosterol, β-sitosterol-3-O-glucosid, α-spinasterol, α-spinasterol-3-O-glucoside
	Centella asiatica	Triterpenes (asiaticoside, asiatic acid, madecassic acid), flavonoids (kaempferol), monoterpenes (camphor), fatty acids (palmitic acid)
	Cnidium monnieri	Monoterpenes (pinene), cnidium lactone
	Saposhnikovia divaricata	Polyacetylenes, furanocoumarins, chromones

Araliaceae	Eleutherococcus senticosus	Saponins (ginsenosides), polyacetylenes, fatty acids, amino acids, polysaccharides
	Panax ginseng China	Saponins (ginsenosides Rb₁, Rb₂, Rc, Rd, Re and Rg₁), polyacetylenes (panaxynol, panaxydol, panaxytriol, falcarindiol), fatty acids, amino acids, polysaccharides
	Panax ginseng Korea	Saponins (ginsenosides Rb₁, Rb₂, Rc, Rd, Re and Rg₁), polyacetylenes (panaxynol, panaxydol, panaxytriol, falcarindiol), fatty acids, amino acids, polysaccharides
	Panax notoginseng	Saponins (ginsenosides Rb₁, Rb₂, Rc, Rd, Re and Rg₁), polyacetylenes (panaxynol, panaxydol, panaxytriol, falcarindiol), fatty acids, amino acids, polysaccharides

Arecaceae	Areca catechu	Alkaloids (arecoline, arecaidin, arecolidin, guvacolin, guvacin)

Asclepiadaceae	Cynanchum paniculatum	Glucosides (cynanchocerin, cynanchin)

Asteraceae	Artemisia annua	Sesquiterpene lactones (artemisinin, arteannuin, artemisitene), monoterpenes (1,8 cineol, borneol, camphor, menthol), coumarins (coumarin, scopoletin)
	Artemisia capillaris	Sesquiterpene lactones, monoterpenes (1,8 cineol, borneol, camphor, menthol), coumarins (coumarin, scopoletin)
	Arctium lappa	Monoterpenes, polyacetylenes (falcarinol), fatty acids, sterols
	Centipeda minima	Monoterpenes (thymol), terpene glycosids, sesquiterpene lactones
	Chrysanthemum indicum	Monoterpenes (1,8-cineole, pinene, borneol, camphor), tannins
	Chrysanthemum morifolium	Monoterpenes (1,8-cineole, pinene, borneol, camphor), tannins
	Eclipta prostrata	Monoterpenes, volatile compounds (Heptadecane, 6,10,14-trimethyl-2-pentadecanone, n-hexadecanoic acid, pentadecane, eudesma-4(14),11-diene, phytol, octadec-9-enoic acid, 1,2-benzenedicarboxylic acid diisooctyl ester, (Z,Z)-9,12-octadecadienoic acid)
	Senecio scandens	Pyrrolizidine alkaloids, terpenoids
	Siegesbeckia orientalis	Phytosterols (β-sitosterol)
	Taraxacum officinale	Sesquiterpene lactones, phenolic acids, triterpene saponins, inulin, phytosterols (β-sitosterol)

Berberidaceae	Berberis bealei	Alkaloids (berberine, columbamine, jatrorrhizine, palmatine)
	Dysosma versipellis	Flavonoids, podophyllotoxin lignans
	Epimedium koreanum	Flavonoids (quercetin, maohuoside B, epimedin A, epimedin B, icariin, icriside II, icariside I, epimedoside A, hexandraside E)

Brassicaceae	Capsella bursa-pastoris	Flavonoids, terpenes, glucosinolates, saponins, tannins
	Isatis indigotica (root)	Flavonoids, glucosinolates, alkaloids (isatisine A, indican, isatin, indirubin and indigotin)
	Isatis indigotica (leaf)	Flavonoids, glucosinolates, alkaloids (isatisine A, indican, isatin, indirubin, indigotin)

Caprifoliaceae	Lonicera confusa	Flavonoids (rutin, quercetin, luteilin-7-O-beta-D-galactoside, lonicerin), chlorogenic acid, beta-sitosterol, tetratriacontane)

Convallariaceae	Polygonatum kingianum	Flavonoids, steroidal saponins

Crassulaceae	Rhodiola rosea	Glucosides (salidroside, tyrosol)

Cupressaceae	Platycladus orientalis	Monoterpenes

Dryopteridaceae	Cyrtomium fortunei	Flavonoids

Ephedraceae	Ephedra sinica	Phenylethylamine alkaloids (ephedrine)

Equisetaceae	Equisetum hiemale	Flavonoids, silicic acids

Euphorbiaceae	Croton tiglium	Glyceryl crotonate, crotonic acid, crotonic resin, phorbol esters (phorbol formate, phorbol butyrate, phorbol crotonate)

Fabaceae	Abrus cantoniensis	Lectins, indolalkaloids
	Acacia catechu	Flavonoids (quercetin, rutin), catechin, epicatechin
	Cassia tora	Flavonoids, dianthrone glycosides (sennoside A, B), anthraquinones (anthrones, emodin, rhein)
	Desmodium styracifolium	Monoterpenes, alkaloids
	Glycyrrhiza inflata	Flavonoids, isoflavonoids, chalcone (liquiritin, isoliquiritin), saponins (glycyrrhizic acid, 4-hydroxy-glycyrrhtinic acid), monoterpenes (1-(2-Furyl)propan-2-one), pyrazine (2-acetyl-1-furfuryl pyrrole), benzene (1-methoxy-4-isopropylbenzene)
	Spatholobus suberectus	Flavonoids, catechin, pyranoside
	Sutherlandia frutescens	Flavonoids, triterpene saponins, L-canavanin, pinitol

Geraniaceae	Geranium wilfordii	Flavonoids, tannins, monoterpenes
	Pelargonium sidoides	Flavonoids, tannins, coumarines, monoterpenes

Ginkgoaceae	Ginkgo biloba	Flavonoids (glycosides of kaempferol, quercetin, isorhamnetin), bisflavonoids, proanthocyanidins, ginkgolic acid, the sesqiterpene alcohol bilobalide, terpene lactones, diterpene lactones (ginkgolides)

Hypericaceae	Hypericum japonicum	Hypericin, hyperforin, monoterpenes, flavonoids, tannins, saponins

Iridaceae	Belamcanda chinensis	Flavonoids (belamcandin, iridin)

Lamiaceae	Mentha haplocalyx	Monoterpenes (menthol)
	Prunella vulgaris	Triterpene saponins, flavonoids (rutin) tannins, rosmarinic acid, monoterpenes (camphor)
	Scutellaria baicalensis	Flavonoids, iridoid glycosides

Lauraceae	Cinnamomum cassia	Monoterpenes (1,8-cineol, pinene, cinnamaldehyde), coumarins, tannins

Loranthaceae	Taxillus chinensis	Flavonoids (avicularin, quercetin)

Lythraceae	Punica granatum	Tannins (punicalin, punicalagin), piperidine alkaloids

Magnoliaceae	Magnolia officinalis	Tannins, flavonoids (rutin), sesquiterpenes, monoterpenes (1,8-cineol)

Melanthiaceae	Paris polyphylla	Steroidal saponins (dioscin, polyphyllin D)

Myrsinaceae	Lysimachia christinae	Flavonoids, tannins, triterpene saponins

Myrtaceae	Eucalyptus robusta	Monoterpenes (1,8-cineol) sesquiterpenes

Ophioglossaceae	Ophioglossum vulgatum	Quercetin 3-O-methyl ether, ophioglonin

Orchidaceae	Dendrobium loddigesii	Alkaloids (dendrobine, nobiline)

Paeoniaceae	Paeonia lactiflora	Flavonoids, (kaempferol), β-sitosterol, resveratrol derivatives, phytoestrogens, monoterpene glycosid (paeoniflorin)

Pedaliaceae	Harpagophytum procumbens	Iridoid glycosides (harpagide, harpagoside)

Poaceae	Cymbopogon distans	Monoterpenes (1,8-cineol, pinene, cymbopogone, cymbopogonol)

Polygonaceae	Fallopia japonica (syn. Polygonum cuspidatum)	Anthraquinones (emodin, rhein, chrysophanol), tetrahydroxystilbene glucosides, steroidal saponins, tannins
	Fallopia multiflora (syn. Polygonum multiflorum)	Flavonoids, tannins
	Polygonum aviculare	Flavonoids, tannins
	Rheum officinale	Flavonoids, tannins, anthraquinone glycosides (emodin, rhein)

Ranunculaceae	Coptis chinensis	Alkaloids (berberine, palmatine, coptisine, columbamine, epiberberine)

Rosaceae	Rosa chinensis	Flavonoids, tannins, carotinoids, vitamin C
	Rosa laevigata	Flavonoids, tannins, carotinoids, vitamin C
	Sanguisorba officinalis	Tannins, flavonoids, saponins, proanthocyanidins

Rubiaceae	Hedyotis diffusa	Iridoid glycosides

Rutaceae	Evodia lepta	Indole alkaloids, (evodiamin, rutecarpin), chromenes
	Evodia rutaecarpa	Indole alkaloids, (evodiamin, rutecarpin)
	Phellodendron chinense	Isoquinoline alkaloids (berberine, palmatine, jatrorrhizine), sesquiterpene lactones

Saururaceae	Houttuynia cordata	Flavonoids (quercetin, quercetin 3-rhamnoside), norcepharadione B

Schisandraceae	Kadsura longipedunculata	Lignans (kadsurilignans), triterpenoid acids, triterpene dilactones, camphene, borneol

Selaginellaceae	Selaginella tamariscina	Flavonoids (amentoflavone, isocryptomerin, biflavonoids), sterols

Valerianaceae	Patrinia scabiosaefolia	Triterpene saponins, iridoid glycosides (patrinoside)

Verbenaceae	Verbena officinalis	Iridoid glycosides, flavonoids

Violaceae	Viola yezoensis	Flavonoids, saponins

Zingiberaceae	Alpinia galanga	Monoterpenes (camphor, cineole, d-pinene, eugenol, cadinene), flavonoids (galangin, riboflavin), niacin, 1′-acetoxychavicol acetate, ascorbic acid
	Alpinia oxyphylla	Monoterpenes (camphor, cineole, d-pinene, eugenol, cadinene), flavonoids (galangin, riboflavin), niacin, 1′-acetoxychavicol acetate, ascorbic acid

Table 3.

Cytotoxicity against cancer cells, Cos7 fibroblasts, and antiviral activity against HBV and flaviviruses of water extracts obtained from 82 medicinal plants.

				Antiumor Effect						Antiviral Effect

Family	Species	IPMB/No.	GenBank	Cos 7 IC₅₀ (μg/mL)	HeLa IC₅₀ (μg/mL)	HepG2 IC₅₀ (μg/mL)	SK-Hep1 IC₅₀ (μg/mL)	LS 174T IC₅₀ (μg/mL)	aanti-BVDV Toxicity on EBTr cells	banti-BVDV protection in EBTr cells	canti-HBV effect in Hep G2 2.2.15	dToxicity at effective doses
Acanthaceae	Andrographis paniculata	P6838/04	JF949965	255.6	576.0	170	80	>200	0	0	++	++

Amaranthaceae	Celosia cristata	P6848/14	JF949970	263.9	2773.5	200	180	>200	0	++	0	ND

Apiaceae	Bupleurum chinense	P6844/10	JF950021	15.6	339.3	120	100	100	++	0	ND	ND
	Bupleurum marginatum	P6845/11	JF949968	350.6	838.1	ND	ND	ND	ND	ND	ND	ND
	Centella asiatica	P6849/15	JF950022	325.8	1436.8	>200	40	70	++++	0	0	ND
	Cnidium monnieri	P6854/20	JF949973	339.7	775.5	>200	>200	>200	++++	0	0	ND
	Saposhnikovia divaricata	P6902/68	JF949988	153.0	1024.7	>200	200	155	++	0	++	++

Araliaceae	Eleutherococcus senticosus	P6919/79	-	130.5	430.0	>200	125	160	++	++	0	ND
	Panax ginseng	P8088/81	JF950028	151.7	2594.6	>200	140	>200	0	+	0	0
	Panax notoginseng	P6887/53	JF950030	182.3	1574.9	>200	>200	200	0	0	ND	ND

Arecaceae	Areca catechu	P6840/06	-	16.6	378.1	40	21	90	++++	0	ND	ND

Asclepiadaceae	Cynanchum paniculatum	P6858/24	JF949975	220.7	693.9	>200	130	>200	++	++	0	ND

Asteraceae	Artemisia annua	P6841/07	JF949966	288.6	775.5	177	50	>200	++++	0	++	++
	Artemisia capillaris	P6842/08	JF949967	201.9	561.7	142	30	>200	++	0	++	++
	Arctium lappa	P6839/05	JF949994	355.5	516.3	200	5	>200	++	0	0	ND
	Centipeda minima	P6850/16	-	55.6	207.2	72	0,5	130	++	++	ND	ND
	Chrysanthemum indicum	P6851/17	JF949971	320.9	583.4	130	8	200	++++	0	0	ND
	Chrysanthemum morifolium	P6852/18	JF949972	760.4	1045.8	>200	180	>200	++	0	0	ND
	Eclipta prostata	P6863/29	JF950000	291.7	667.0	>200	30	120	++	++	0	ND
	Senecio scandens	P6905/71	JF949989	114.2	607.9	3.5	50	25	++++	++	ND	ND
	Siegesbeckia orientalis	P6906/72	JF949990	159.2	542.8	40	100	50	++	++	ND	ND
	Taraxacum officinale	P6908/74	JF950019	156.9	708.5	130	147	65	++	++	0	ND

Berberidaceae	Berberis bealei	P6883/49	JF949996	270.0	659.4	63	60	70	ND	ND	ND	ND
	Dysosma versipellis	P6862/28	-	1276.9	1274.8	>200	3.5	3.5	++++	0	0	ND
	Epimedium koreanum	P6865/31	JF950002	140.7	280.2	5	32	10	++	0	ND	ND

Brassicaceae	Isatis indigotica (root)	P6877/43	JF949981	557.2	2427.4	>200	> 200	>200	0	0	0	ND
	Isatis indigotica (leaf)	P6878/44	JF949981	93.5	1223.5	170	98	80	++	++	0	ND

Caprifoliaceae	Lonicera confusa	P6880/46	JF949982	446.8	812.2	>200	>200	>200	++	0	0	ND

Convallariaceae	Polygonatum kingianum	P6892/58	JF950027	298.4	2321.7	>200	130	42	++	0	0	ND

Crassulaceae	Rhodiola rosea	P6920/84	-	61.9	144.4	160	110	40	++	++	0	ND

Cupressaceae	Platycladus orientalis	P6891/57	JF950011	97.7	428.2	>200	155	10	++	0	0	ND

Dryopteridaceae	Cyrtomium fortunei	P6859/25	JF949998	30.4	567.4	>200	>200	>200	++	++	0	ND

Ephedraceae	Ephedra sinica	P6864/30	JF950001	69.1	193.1	200	150	>200	++++	++	ND	ND

Equisetaceae	Equisetum hiemale	P6866/32	JF950003	265.9	1058.2	5	>200	>200	++	0	ND	ND

Euphorbiaceae	Croton tiglium	P6856/22	-	166.2	1052.7	140	50	>200	++++	0	++	++

Fabaceae	Abrus cantoniensis	P6835/01	JF949964	575.2	587.1	>200	100	>200	++	++	0	ND
	Acacia catechu	P6836/02	-	35.7	157.5	>200	25	>200	++	++	++	++
	Cassia tora	P6847/13	JF949969	481.3	1519.3	>200	0.5	>200	0	+	++	++
	Desmodium styracifolium	P6861/27	JF949976	333.5	651.4	>200	>200	150	0	0	0	ND
	Glycyrrhiza inflata	P6873/39	JF950025	583.9	2288.0	>200	>200	185	0	0	++++	0
	Spatholobus suberectus	P6907/73	JF949991	16.6	174.1	100	135	70	++	0	ND	ND
	Sutherlandia frutescens	tba/83	-	857.6	1670.7	>200	70	>200	0	0	0	ND

Geraniaceae	Geranium wilfordii	P6867/33	JF949977	225.8	62.1	80	45	200	++	++	ND	ND
	Pelargonium sidoides	tba/82	-	15.2	62.2	200	>200	45	++	0	0	ND

Ginkgoaceae	Ginkgo biloba	P6872/38	JF950005	450.8	1717.0	> 200	9	>200	0	+	0	ND

Hypericaceae	Hypericum japonicum	P6876/42	JF949980	151.8	445.5	165	80	100	++	++	0	ND

Iridaceae	Belamcanda chinensis	P6843/09	JF949995	222.1	1378.8	>200	>200	>200	++	0	++	++

Lamiaceae	Mentha haplocalyx	P6884/50	JF949984	285.7	519.1	70	82	>200	++++	0	ND	ND
	Prunella vulgaris	P6896/62	JF950013	21.5	341.3	100	145	80	++	0	ND	ND
	Scutellaria baicalensis	P6903/69	JF950017	46.4	150.0	80	50	120	++	0	ND	ND

Lauraceae	Cinnamomum cassia	P6853/19	JF950023	453.9	713.6	180	>200	>200	++++	0	++	++

Loranthaceae	Taxillus chinensis	P6909/75	JF949992	181.7	1023.2	>200	>200	155	0	0	0	ND

Lythraceae	Punica granatum	P6897/63	JF950014	8.6	152.4	100	100	60	++++	0	ND	ND

Magnoliaceae	Magnolia officinalis	P6882/48	JF950008	73.0	451.5	ND	ND	ND	ND	ND	ND	ND

Melanthiaceae	Paris polyphylla	P6888/54	JF950010	38.4	42.6	54	168	8	++++	++	ND	ND

Myrsinaceae	Lysimachia christinae	P6881/47	JF949983	152.1	431.4	>200	>200	>200	++	0	0	ND

Myrtaceae	Eucalyptus robusta	P6868/34	-	94.1	15.8	ND	ND	ND	ND	ND	ND	ND

Ophioglossaceae	Ophioglossum vulgatum	P6885/51	JF950009	344.0	1780.1	>200	>200	>200	0	++	0	ND

Orchidaceae	Dendrobium loddigesii	P6860/26	JF949999	104.0	294.4	>200	70	160	++	++	0	ND

Paeoniaceae	Paeonia lactiflora	P6886/52	JF950026	148.2	287.3	>200	>200	10	0	0	ND	ND

Pedaliaceae	Harpagophytum procumbens	tba/80	-	242.9	733.4	160	190	100	++	++	0	ND

Poaceae	Cymbopogon distans	P6857/23	JF949974	257.7	486.1	>200	>200	>200	++++	0	++	++

Polygonaceae	Fallopia japonica	P6894/60	JF950004	39.8	596.4	>200	>200	80	++++	++	0	ND
	Polygonum aviculare	P6893/59	JF950012	82.6	488.6	>200	>200	10	++	0	0	ND
	Polygonum multiflorum	P6895/61	JF949987	61.3	928.0	ND	ND	ND	ND	ND	ND	ND
	Rheum officinale	P6898/64	JF950015	51.5	670.9	200	25	200	++	0	0	ND

Ranunculaceae	Coptis chinensis	P6855/21	JF950024	118.3	101.0	10	2	18	++++	0	ND	ND

Rosaceae	Rosa chinensis	P6899/65	-	24.3	135.8	ND	ND	ND	ND	ND	ND	ND
	Rosa laevigata	P6900/66	-	93.6	781.7	190	135	60	++	++	0	ND
	Sanguisorba officinalis	P6901/67	JF950016	20.5	87.0	ND	ND	ND	ND	ND	ND	ND

Rubiaceae	Hedyotis diffusa	P6874/40	JF949979	158.7	1542.7	>200	>200	5	++	++	++++	0

Rutaceae	Evodia lepta	P6869/35	JF949978	419.2	971.0	>200	20	>200	++++	0	++++	0
	Evodia rutaecarpa	P6870/36	-	1176.9	185.6	>200	25	100	++++	++	0	ND
	Phellodendron chinense	P6890/56	JF949986	282.9	750.3	50	10	85	++	++	ND	ND

Saururaceae	Houttuynia cordata	P6875/41	JF950006	633.2	2835.9	>200	135	5	0	++	0	ND

Schisandraceae	Kadsura longipedunculata	P6879/45	JF950007	6.8	167.6	>200	20	20	++	++	0	ND

Selaginellaceae	Selaginella tamariscina	P6904/70	JF950018	103.9	703.4	>200	>200	200	0	++	0	0

Valerianaceae	Patrinia scabiosaefolia	P6889/55	JF949985	147.3	525.5	168	87	35	++	++	0	ND

Verbenaceae	Verbena officinalis	P6910/76	JF950020	93.9	416.9	100	168	117	++	0	ND	ND

Violaceae	Viola yezoensis	P6911/77	JF949993	135.0	1459.2	170	200	140	0	+	ND	ND

Zingiberaceae	Alpinia galanga	P6837/03	-	952.8	2357.3	>200	>200	>200	0	++	++	++
	Alpinia oxyphylla	P6917/78	-	105.8	1802.2	>200	>200	155	0	++	0	ND

Toxicity on EBTr cells: 0, not toxic; ++, toxic at high concentrations; ++++, toxic in all concentrations;

Anti-BVDV protection in EBTr cells: 0, without effect; +, protection at high concentrations; ++, protection at low concentrations;

Anti-HBV effect in HepG2 2.2.15 cells: 0, without effect; ++, effect comparable to toxicity; ++++, high ability to reduce HBV-DNA;

Toxicity at effective dose on HepG2 2.2.15 cells: 0, not toxic; ++, effect comparable to reduction in HBV DNA. ND: Not determined.

Table 4.

Cytotoxicity against HeLa cancer cells, Cos7 fibroblasts and Trypanosoma brucei brucei of organic extracts obtained from 82 medicinal plants.

Family	Species	IPMB/No.	GenBank	CH₂Cl₂	CH₂Cl₂	CH₂Cl₂	RatioHeLa/T. b. brucei	RatioCos7/T. b. brucei	MeOH	MeOH	MeOH	RatioHeLa/T. b. brucei	RatioCos7/T. b. brucei

				HeLa	Cos 7	T. b. brucei			HeLa	Cos 7	T. b. brucei
Acanthaceae	Andrographis paniculata	P6838/04	JF949965	188.4	104.7	16.8	11	6	323.3	344.7	28.8	11	12

Amaranthaceae	Celosia cristata	P6848/14	JF949970	472.0	136.0	55.2	9	2	499.8	28.4	77.2	6	0.3

Apiaceae	Bupleurum chinense	P6844/10	JF950021	235.2	87.1	17.0	14	5	646.4	358.7	120.8	5	3
	Bupleurum marginatum	P6845/11	JF949968	176.0	67.4	16.2	11	4	1147.0	576.0	111.9	10	5
	Centella asiatica	P6849/15	JF950022	175.0	64.9	14.0	13	4	773.0	392.8	44.7	17	8
	Cnidium monnieri	P6854/20	JF949973	127.1	37.0	14.9	9	2	251.1	120.0	17.9	14	6
	Saposhnikovia divaricata	P6902/68	JF949988	410.1	45.9	5.1	80	9	1515.6	1575.4	999.5	2	1

Araliaceae	Eleutherococcus senticosus	P6919/79	-	300.0	61.4	13.5	22	4	692.0	190.1	17.3	40	11
	Panax ginseng	P8088/81	JF950028	152.4	47.7	0.9	169	53	1427.9	510.8	319.0	4	1
	Panax notoginseng	P6887/53	JF950030	263.0	6.4	0.9	292	7	1241.6	229.5	469.6	2	0.4

Arecaceae	Areca catechu	P6840/06	-	1023.3	117.0	22.5	45	5	414.2	31.0	118.1	4	0.2

Asclepiadaceae	Cynanchum paniculatum	P6858/24	JF949975	395.6	114.2	53.1	7	2	500.5	227.7	39.3	13	5

Asteraceae	Artemisia annua	P6841/07	JF949966	107.9	34.5	8.1	13	4	287.2	201.1	51.2	6	4
	Artemisia capillaris	P6842/08	JF949967	93.5	29.4	10.6	9	3	314.9	215.4	51.9	6	4
	Arctium lappa	P6839/05	JF949994	345.0	344.2	3.6	96	96	1467.7	1813.0	2229.0	0.7	0.8
	Centipeda minima	P6850/16	-	63.3	10.4	2.2	29	5	219.1	54.2	13.3	16	4.0
	Chrysanthemum indicum	P6851/17	JF949971	152.1	63.5	16.0	10	4	355.7	287.2	15.3	23	18
	Chrysanthemum morifolium	P6852/18	JF949972	129.4	42.8	19.3	7	2	349.2	166.7	24.9	14	6
	Eclipta prostata	P6863/29	JF950000	266.4	112.0	38.1	7	3	329.7	186.1	39.6	8	4.6
	Senecio scandens	P6905/71	JF949989	268.6	143.5	13.1	21	11	299.3	126.2	18.6	16	6
	Siegesbeckia orientalis	P6906/72	JF949990	101.5	17.7	7.9	13	2	237.5	84.4	12.3	19	6
	Taraxacum officinale	P6908/74	JF950019	232.8	177.1	17.5	13	10	636.7	485.3	64.9	10	7

Berberidaceae	Berberis bealei	P6883/49	JF949996	93.8	13.3	5.9	16	2	149.7	35.3	7.8	19	4
	Dysosma versipellis	P6862/28	-	213.9	49.9	39.5	5	1	385.2	54.9	53.2	7	1.0
	Epimedium koreanum	P6865/31	JF950002	48.7	3.5	4.2	12	0.8	257.5	30.7	12.6	20	2

Brassicaceae	Isatis indigotica (root)	P6877/43	JF949981	196.4	42.3	2.9	68	14	674.3	324.4	94.6	7	3
	Isatis indigotica (leaf)	P6878/44	JF949981	321.5	0.6	45.3	7	0.01	274.2	90.6	14.6	19	6

Caprifoliaceae	Lonicera confusa	P6880/46	JF949982	226.5	58.9	16.2	14	3	923.5	118.9	38.0	24	3

Convallariaceae	Polygonatum kingianum	P6892/58	JF950027	279.6	53.9	52.6	5	1	1517.9	1535.3	119.5	13	12

Crassulaceae	Rhodiola rosea	P6920/84	-	164.1	74.6	43.9	4	1	-	87.4	-	-	-

Cupressaceae	Platycladus orientalis	P6891/57	JF950011	121.7	21.8	17.7	7	1	705.5	158.2	84.2	8	1

Dryopteridaceae	Cyrtomium fortunei	P6859/25	JF949998	572.4	132.1	37.1	15	3	722.0	348.7	61.0	12	5

Ephedraceae	Ephedra sinica	P6864/30	JF950001	95.3	41.8	20.9	5	2	163.5	36.7	23.4	7	1

Equisetaceae	Equisetum hiemale	P6866/32	JF950003	125.6	35.7	30.9	4	1	241.2	243.5	51.6	4	4

Euphorbiaceae	Croton tiglium	P6856/22	-	422.9	225.9	86.5	5	2	297.0	222.1	150.4	2	1

Fabaceae	Abrus cantoniensis	P6835/01	JF949964	494.4	129.4	14.5	34	9	612.4	733.1	73.5	8	10
	Acacia catechu	P6836/02	-	164.1	31.5	13.1	12	2	318.0	34.8	50.8	6	0.6
	Cassia tora	P6847/13	JF949969	1335.4	189.1	185.9	7	1	670.9	75.9	276.9	2	0.2
	Desmodium styracifolium	P6861/27	JF949976	156.0	139.8	16.3	10	8	324.3	104.1	40.1	8	2
	Glycyrrhiza inflata	P6873/39	JF950025	26.4	6.9	6.4	4	1	528.3	126.8	39.0	14	3
	Spatholobus suberectus	P6907/73	JF949991	299.1	154.6	25.4	12	6	237.5	54.8	67.8	4	0.8
	Sutherlandia frutescens	tba/83	-	367.7	259.3	41.8	9	6	586.6	352.0	87.4	7	4

Geraniaceae	Geranium wilfordii	P6867/33	JF949977	99.1	17.0	23.0	4	0.7	236.0	169.8	13.3	18	12
	Pelargonium sidoides	tba/82	-	488.2	218.2	52.1	9	4	112.3	95.7	18.3	6	5

Ginkgoaceae	Ginkgo biloba	P6872/38	JF950005	768.3	15.3	71.9	11	0.2	302.9	260.1	39.3	8	6

Hypericaceae	Hypericum japonicum	P6876/42	JF949980	163.3	10.8	21.3	8	0.5	177.5	100.9	23.6	8	4

Iridaceae	Belamcanda chinensis	P6843/09	JF949995	324.4	89.2	22.3	15	4	522.6	319.5	80.2	7	4

Lamiaceae	Mentha haplocalyx	P6884/50	JF949984	108.5	34.1	14.7	7	2	375.0	147.8	16.2	23	9
	Prunella vulgaris	P6896/62	JF950013	282.1	90.4	13.2	21	7	475.4	494.5	25.1	19	19
	Scutellaria baicalensis	P6903/69	JF950017	90.9	287.9	7.4	12	39	367.6	28.8	86.2	4	0.3

Lauraceae	Cinnamomum cassia	P6853/19	JF950023	138.9	23.2	11.0	13	2	272.4	108.4	13.4	20	8

Loranthaceae	Taxillus chinensis	P6909/75	JF949992	417.8	68.6	27.2	15	2	1213.4	378.2	59.2	20	6

Lythraceae	Punica granatum	P6897/63	JF950014	583.3	126.6	14.6	40	8	211.2	218.6	8.1	26	27

Magnoliaceae	Magnolia officinalis	P6882/48	JF950008	23.6	5.4	0.9	26	6	49.1	13.1	4.3	11	3

Melanthiaceae	Paris polyphylla	P6888/54	JF950010	952.6	24.0	73.6	13	0.3	35.0	5.5	11.8	3	0.4

Myrsinaceae	Lysimachia christinae	P6881/47	JF949983	53.4	137.3	20.6	3	7	1752.6	436.3	52.1	34	8

Myrtaceae	Eucalyptus robusta	P6868/34	-	-	-	-			181.4	15.2	16.3	11	1

Ophioglossaceae	Ophioglossum vulgatum	P6885/51	JF950009	188.9	62.8	19.8	10	3	469.0	68.8	33.2	14	2

Orchidaceae	Dendrobium loddigesii	P6860/26	JF949999	83.0	25.7	13.5	6	2	232.8	61.6	27.6	8	2

Paeoniaceae	Paeonia lactiflora	P6886/52	JF950026	166.9	34.0	9.1	18	3	294.6	309.8	11.7	25	26

Pedaliaceae	Harpagophytum procumbens	tba/80	-	36.2	15.8	0.9	40	17	692.6	217.2	21.4	32	10

Poaceae	Cymbopogon distans	P6857/23	JF949974	425.9	114.5	31.1	14	3	98.8	17.6	18.9	5	1

Polygonaceae	Fallopia japonica	P6894/60	JF950004	88.0	2.8	13.1	7	0.2	317.3	19.5	19.0	17	1
	Polygonum aviculare	P6893/59	JF950012	118.5	53.3	18.2	7	3	342.3	226.5	49.1	7	4
	Polygonum multiflorum	P6895/61	JF949987	469.4	107.7	98.6	5	1	437.4	48.8	62.1	7	0.7
	Rheum officinale	P6898/64	JF950015	22.5	-	34.0	0.6	-	270.9	35.3	24.5	11	1

Ranunculaceae	Coptis chinensis	P6855/21	JF950024	100.0	39.5	12.9	8	3	81.8	3.7	0.4	205	9

Rosaceae	Rosa chinensis	P6899/65	-	559.4	141.5	20.1	28	7	266.6	36.7	12.5	21	3
	Rosa laevigata	P6900/66	-	712.3	151.8	20.6	35	7	1855.4	1100.1	102.9	18	10
	Sanguisorba officinalis	P6901/67	JF950016	66.5	26.7	12.3	5	2	158.5	41.6	4.0	40	10

Rubiaceae	Hedyotis diffusa	P6874/40	JF949979	147.8	45.3	13.3	11	3	796.1	418.1	24.9	32	16

Rutaceae	Evodia lepta	P6869/35	JF949978	232.0	42.0	13.9	17	3	350.7	427.7	44.4	8	9
	Evodia rutaecarpa	P6870/36	-	50.4	8.7	16.8	3	0.5	297.4	178.5	29.5	10	6
	Phellodendron chinense	P6890/56	JF949986	370.1	71.3	15.6	24	4	487.6	85.5	14.1	35	6

Saururaceae	Houttuynia cordata	P6875/41	JF950006	279.9	48.2	68.3	4	0.7	575.2	63.9	97.6	6	0.6

Schisandraceae	Kadsura longipedunculata	P6879/45	JF950007	9.9	1.8	0.1	99	18	86.1	43.9	11.8	7	3

Selaginellaceae	Selaginella tamariscina	P6904/70	JF950018	339.2	98.8	13.6	25	7	393.9	150.9	33.4	12	4

Valerianaceae	Patrinia scabiosaefolia	P6889/55	JF949985	140.5	38.7	13.7	10	3	159.4	15.9	19.0	8	0.8

Verbenaceae	Verbena officinalis	P6910/76	JF950020	298.1	145.9	16.5	18	9	334.7	37.7	20.5	16	1

Violaceae	Viola yezoensis	P6911/77	JF949993	59.6	60.8	3.3	18	18	297.5	19.1	24.7	12	0.7

Zingiberaceae	Alpinia galanga	P6837/03	-	55.7	5.7	1.4	39	4	111.7	53.4	15.4	7	3
	Alpinia oxyphylla	P6917/78	-	119.6	30.4	0.7	170	43	213.8	110.2	2.0	107	55

Financial Support: This study was supported in part by the Ministerio de Ciencia y Tecnología, Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica (Grant SAF2010-15517), Spain.

Appendix

Origin and current area of use of the medicinal plants included in our study.

Family	Species	Origin	Area of Use
Acanthaceae	Andrographis paniculata	India	India, Sri Lanka, SE Asia, East Asia

Amaranthaceae	Celosia cristata	Tropical Asia	India, SE Asia, China, Africa, South America

Apiaceae	Bupleurum chinense	China	East Asia, China
	Bupleurum marginatum	China	East Asia, China
	Centella asiatica	East Asia, India, Sri Lanka, northern Australia, Iran, Melanesia, Papua New Guinea	East Asia, India, Sri Lanka, Australia, Melanesia, Papua New Guinea, Middle East, Africa
	Cnidium monnieri	China	East Asia, China
	Saposhnikovia divaricata	Central Asia (steppe region)	Central Asia, East Asia, China

Araliaceae	Eleutherococcus senticosus	Siberia	Siberia, China, Korea, Japan
	Panax ginseng China	China	Siberia, China, Korea, Japan
	Panax ginseng Korea	Korea	Siberia, China, Korea, Japan
	Panax notoginseng	China	Siberia, China, Korea, Japan

Arecaceae	Areca catechu	Malaysia, Philipines	SE Asia, East Asia, India, Sri Lanka, Papua New Guinea

Asclepiadaceae	Cynanchum paniculatum	SE Asia	East Asia, SE Asia

Asteraceae	Artemisia annua	Asia, introduced worldwide	worldwide
	Artemisia capillaris	Asia	Asia
	Arctium lappa	Northern Hemisphere (Europe, Asia, North America)	Europe, Asia
	Centipeda minima	Asia, Himalaya	Asia
	Chrysanthemum indicum	India	Asia
	Chrysanthemum morifolium	Asia	Asia
	Eclipta prostrata	Tropical Asia, South America	Tropical Asia, East Asia, South America
	Senecio scandens	Asia	Asia
	Siegesbeckia orientalis	Tropical Asia	Tropical Asia, East Asia, Africa
	Taraxacum officinale	Northern Hemisphere (Europe, Asia, North America)	Europe, Asia, North America

Berberidaceae	Berberis bealei	Asia, introduced in America, Europe	Asia, America, Europe
	Dysosma versipellis	East Asia	East Asia, China
	Epimedium koreanum	East Asia (Korea)	East Asia, China

Brassicaceae	Capsella bursa-pastoris	Northern Hemisphere (Europe, Asia, North America)	Europe, Asia
	Isatis indigotica (root)	Central Asia (steppe region)	Central Asia, East Asia, China
	Isatis indigotica (leaf)	Central Asia (steppe region)	Central Asia, East Asia, China

Caprifoliaceae	Lonicera confusa	East Asia	East Asia, China

Convallariaceae	Polygonatum kingianum	Asia	Asia

Crassulaceae	Rhodiola rosea	Northern Hemisphere (Europe, Asia, North America)	Europe, Asia, North America

Cupressaceae	Platycladus orientalis	China, introduced in most of Asia	Asia

Dryopteridaceae	Cyrtomium fortunei	Asia, introduced in America, Europe	Asia

Ephedraceae	Ephedra sinica	China	East Asia

Equisetaceae	Equisetum hiemale	Northern Hemisphere (Europe, Asia, North America)	Europe, Asia, North America

Euphorbiaceae	Croton tiglium	SE Asia	East Asia, SE Asia

Fabaceae	Abrus cantoniensis	Southern China	East Asia, SE Asia
	Acacia catechu	East Asia, SE Asia	East Asia, SE Asia
	Cassia tora	East Asia, SE Asia, introduced to Middle and South America, Africa, Middle East	Europe, Asia, America, Africa
	Desmodium styracifolium	SE Asia	East Asia, SE Asia
	Glycyrrhiza inflata	Central Asia (Mongolia, China)	East Asia, Central Asia
	Spatholobus suberectus	Tropical Asia	India, East Asia, SE Asia
	Sutherlandia frutescens	South Africa	South Africa, Europe

Geraniaceae	Geranium wilfordii	East Asia	East Asia, China
	Pelargonium sidoides	South Africa	South Africa, Europe

Ginkgoaceae	Ginkgo biloba	China	Asia, Europe, North America

Hypericaceae	Hypericum japonicum	Japan	East Asia, China

Iridaceae	Belamcanda chinensis	China	East Asia, China

Lamiaceae	Mentha haplocalyx	China	East Asia, China
	Prunella vulgaris	Northern Hemisphere (Europe, Asia, North America)	Europe, Asia, North America
	Scutellaria baicalensis	Central Asia (Russia, Mongolia, China)	East Asia, Central Asia

Lauraceae	Cinnamomum cassia	Tropical Asia (India, East Asia, SE Asia)	India, East Asia, SE Asia

Loranthaceae	Taxillus chinensis	China	East Asia, China

Lythraceae	Punica granatum	Middle East, Himalaya	Europe, Asia, America, Africa

Magnoliaceae	Magnolia officinalis	Himalaya, China	East Asia, China

Melanthiaceae	Paris polyphylla	Himalaya, China	East Asia, China

Myrsinaceae	Lysimachia christinae	China	East Asia, China

Myrtaceae	Eucalyptus robusta	East Australia	Europe, Asia, America, Africa, Australia

Ophioglossaceae	Ophioglossum vulgatum	Northern Hemisphere (Europe, Asia, North America)	Europe, Asia, North America

Orchidaceae	Dendrobium loddigesii	SE Asia	East Asia, SE Asia

Paeoniaceae	Paeonia lactiflora	China	East Asia

Pedaliaceae	Harpagophytum procumbens	South Africa	South Africa, Europe

Poaceae	Cymbopogon distans	Himalaya, China	East Asia, China

Polygonaceae	Fallopia japonica (syn. Polygonum cuspidatum)	East Asia	East Asia, China
	Fallopia multiflora (syn. Polygonum multiflorum)	East Asia	East Asia, China
	Polygonum aviculare	Northern Hemisphere (Europe, Asia, North America)	Europe, Asia, North America
	Rheum officinale	Asia	Europe, Asia, North America

Ranunculaceae	Coptis chinensis	China	East Asia, China

Rosaceae	Rosa chinensis	China	East Asia, China
	Rosa laevigata	SE Asia, China	Europe, Asia, North America
	Sanguisorba officinalis	Northern Hemisphere (Europe, Asia, North America)	Europe, Asia, North America

Rubiaceae	Hedyotis diffusa	East Asia	East Asia, China

Rutaceae	Evodia lepta	East Asia	East Asia, China
	Evodia rutaecarpa	East Asia	East Asia, China
	Phellodendron chinense	Himalaya, China	East Asia, China

Saururaceae	Houttuynia cordata	East Asia, SE Asia	East Asia, SE Asia

Schisandraceae	Kadsura longipedunculata	East Asia	East Asia, China

Selaginellaceae	Selaginella tamariscina	East Asia	East Asia, China

Valerianaceae	Patrinia scabiosaefolia	East Asia	East Asia, China

Verbenaceae	Verbena officinalis	Europe	Europe, Asia, North America

Violaceae	Viola yezoensis	East Asia	East Asia, China

Zingiberaceae	Alpinia galanga	SE Asia	East Asia, SE Asia
	Alpinia oxyphylla	SE Asia	East Asia, SE Asia

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